Locus-specific Imaging of Dynamic Histone Methylations during Reprogramming Reprogramming fibroblasts into induced pluripotent stem cells (iPSCs) represents a revolutionary advancement in the understanding of how specific gene regulations can guide the life of a cell. Epigenetic modifications including chromatin remodeling are early events during the reprogramming process. Histone methylation at different residues can recruit differential sets of chromatin remodeling complexes to regulate chromatin structures and silence/activate gene expressions accordingly. These histone methylations and their combinations at different genomic loci can serve as codes to determine the overall gene expression profile and phenotypic outcomes. However, it is still not understood how histone methylations at specific loci are dynamically regulated during the reprogramming processes in which cells undergo a highly heterogeneous modulation at single cell levels. In this proposal, we will harness the power of directed evolution and high-throughput screening method to systematically develop specific/sensitive FRET (fluorescence resonance energy transfer) biosensors for the monitoring of crucial histone methylations in single cells. We will further develop biosensors with distinct and orthogonal FRET pairs that can simultaneously monitor two different histone methylations in a single live cell for the production of high-resolution images of multiplex epigenetic landscapes. These multiplex histone methylations obtained from individual cells during reprogramming will then be analyzed and integrated together to construct the dynamic histone methylation landscapes. These epigenetic modulations will also be visualized at specific loci to assign the corresponding genomic addresses on the evolving landscape of histone methylations. Established fluorescence markers of cell fate will further be applied to determine how histone methylation codes are coordinated for the regulation of reprogramming. As such, the success of the project should have transformative impact in the field of epigenetics and genetics at single cell levels, particularly related to stem cell reprogramming. Three specific aims are accordingly proposed: Aim 1. Develop high-throughput screening methods for the engineering of FRET biosensors to monitor various histone methylations; Aim 2. Engineer FRET biosensors with distinct colors to monitor the evolving multiplex landscape of histone methylations during reprogramming; Aim 3. Unravel the evolving histone methylation landscapes at specific loci during reprogramming. While the focus of this proposal is to develop tools targeting histone methylations at specific loci and reprogramming outcomes, the strategies and approaches can be extended to monitor, in principle, any epigenetic modification in single cells, including but not limited to histone acetylation and phosphorylation. The developed biosensors for single cell imaging of epigenetic landscape evolution should offer powerful tools for life science, biomedical research, and tissue engineering in general. The results from this project can also lead directly to the dynamic nuclear atlas illustrating how specific histone codes are encrypted in an integrative manner for the regulation of life.